The goal of this research was to identify the causal variant associated with a haplotype affecting fertility in Ayrshire cattle. The US dairy population is routinely monitored to identify cases where homozygotes for a minor allele are expected, but never observed, which can indicate the presence of a genetic defect causing embryonic death. Such a haplotype was identified on chromosome 17 in January 2013. A second haplotype reached the threshold for statistical significance (7 expected homozygotes and none observed) in October 2013. Sire conception rate was 6.1% lower for matings of carrier sires to cows with carrier maternal grandsires, but the effect was not significant. As of December 2016, 23.5 homozygotes were expected but none had been observed. Most carriers trace back to OAK-RIDGE FLASHY KELLOGG (AYUSA000000125168), born in 1961, but many Canadian carriers only trace back as far as WOODLAND VIEW PARDNER ET (AYCAN000000811799), born in 1994. These bulls share an ungenotyped ancestor in OAK-RIDGE LIGHTNING (AYUSA000000120135), born in 1958. This haplotype has been designated Ayrshire haplotype 2 (AH2), and its frequency has increased from 6% in animals born in 1990 to 21.7% for animals born since 2008. Whole-genome sequence data ranging from 9 × to 13 × read-depth for 8 Ayrshire bulls, 3 carriers and 5 non-carriers, was used to search for the causal variant. The analysis used a short-read sequence data analysis pipeline including BWA (v. 0.7.10) and Samtools (v. 1.3). Likely false positive variants and variants with small predicted functional effects were removed after annotation with SNPeFF (v. 4.3). A splice acceptor variant at 51,267,548 bp in the RNA Polymerase 2 Associated Protein (RPAP2) gene was the most likely causal variant in the haplotype. RPAP2 is an essential component of the RNA polymerase 2 holoenzyme necessary for transcription of snRNA species. Experiments with mouse knockouts also found a deficiency of homozygotes, suggesting that RPAP2 is necessary for embryonic development. These findings provide strong evidence for the existence of a new, lethal recessive in Ayrshire cattle. AH2 carrier status should be reported to the industry routinely and its effect on fertility confirmed.